臘狀芽孢桿菌C1L 菌株在臺灣百合根部群聚能力之探討

Studies on the colonization of Bacillus cereus C1L in the roots of Lilium formosanum

林玉儒(Yu-Ju Lin)

指導教授 ： 陳昭瑩

國立臺灣大學/生物資源暨農學院/植物病理與微生物學研究所/碩士(2008年)

https://doi.org/10.6342/NTU.2008.01109

摘要

百合灰黴病(Lily gray mold)為臺灣百合之真菌性病害，由灰黴病菌(Botrytis elliptica (Berk.) Cooke)感染造成葉片焦枯、花器畸形及花瓣萎凋，對臺灣百合之園藝栽作影響甚鉅。為利用生物防治法解決灰黴病菌危害的問題，乃自臺灣百合根圈篩選具有生物防治潛力的菌株，其中以臘狀芽孢桿菌C1L菌株之表現最佳。為便於剖析C1L菌株之群聚 (colonization) 特性，利用對立復黴素(rifampicin)具抗性的C1L菌株，處理於臺灣百合幼苗根圈後，回收根內及根表的細菌，計算族群密度，發現C1L菌株在根表及根內有良好的群聚能力。利用源自臘狀芽孢桿菌幾丁質分解酵素基因(chiCH)之啟動子及pHY300PLK建構可表現綠色螢光蛋白的載體系統，使C1L菌株產生自發性螢光。經綠色螢光標定之C1L菌株，其誘導植株系統性抗病能力(induced systemic resistance)、抑制真菌孢子發芽、菌絲生長的能力及泳動性(motility)皆與野生菌株之功效相近。利用共軛焦雷射顯微鏡觀察螢光標定之C1L菌株在臺灣百合幼苗根部群聚的情形，發現C1L菌株可附著根毛進入根部表層細胞間隙，並進入根部組織之深層細胞間隙。進一步利用轉位誘變之方式，獲得340個C1L菌株之突變株，針對突變株在玉米根內的群聚能力及泳動性進行篩選，經由單邊側翼序列比對方式，發現失去群聚能力之突變株被破壞之基因可能解碼nitric oxide dioxygenase cAMP-dependent protein kinase、N-acetylgalactosamine-6-phosphate deacetylase、glucose-specific IIABC component、replication protein等，而失去泳動性之突變株被破壞的基因可能解碼DNA binding protein、hypothetical protein以及possible collagen-like protein integrase。未來，期望進一步分析C1L菌株在植物根部群聚的模式。

關鍵字

臘狀芽孢桿菌C1L 菌株 ； 臺灣百合 ； 生物防治 ； 群聚作用

並列摘要

Gray mold disease, caused by Botrytis elliptica (Berk.) Cooke, is one of the most important fungal diseases in Lilium formosanum that causes severe losses of yield and quality of lilies. In general, field management by application of fungicides to prevent and decrease disease incidence frequently results in the evolvement of fungicide-resistant strains. Therefore, alternative control measures such as biological control are developed. A biocontrol strain of Bacillus cereus C1L had been screened from the rhizosphere of L. formosanum and it could suppress the disease severity of lily gray mold. In this study, fluorescent C1L strain was constructed by using a promoter driving the expression of chitinase CH of Bacillus cereus and a shuttle vector pHY300PLK to express green fluorescence protein (GFP) in both Escherichia coli and Bacillus cereus. The tests on the abilities of induced systemic resistance (ISR), inhibition of spore germination and hyphal growth, and the bacterial motility indicated that the GFP-labeled C1L strain was similar to the wild-type strain. Examination by confocal laser scanning microscopy showed the presence of GFP-labeled C1L bacterial cells inner the roots of L. formosanum seedlings. The C1L strain could adhere to the root hairs, enter the intercellular space of root surface layer, and present in the in-depth intercellular space of root tissues, indicating that this bacterium could be an epiphyte and endophyte in the plant roots. In order to identify the genes of B. cereus C1L involved in the root colonization, a transposon insertion mutant library including 340 mutant strains was constructed. These mutants were screened for motility and colonization ability within corn roots. The genes identified from mutants of losing colonization ability were predicted to encode nitric oxide dioxygenase cAMP-dependent protein kinase, N-acetylgalactosamine-6-phosphate deacetylase, glucose-specific IIABC component, replication protein…et al. The genes possibly involved in motility were predicted to encode DNA binding protein, hypothetical protein, and possible collagen-like protein integrase. A build-up of model for the colonization of strain C1L in the roots of L. formosanum is the future prospect.

並列關鍵字

Bacillus cereus C1L ； Lilium formosanum ； biological control ； colonization

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